Cerebral malaria (CM) pathogenesis is due to a complex interaction between the vascular system, the immune response, and parasite sequestration. We have reported that administration of compounds that restore NO bioavailability (nitric oxide [NO] donor, NO gas, arginine and citrulline) protect all recipients fom experimental CM (ECM); NO gas and arginine are currently undergoing clinical trials as adjunctive therapy for CM. There are, however, no biomarkers to gauge whether the adjunctive therapy is restoring NO bioavailability. We propose that RBC-derived microparticles (RMPs) play a critical role in reducing NO bioavailability during eCM and hence mediating pathogenesis. While free hemoglobin (Hb) is rapidly degraded, Hb contained within RMPs circulates for extended periods of time and has a 1,000-fold greater NO scavenging than intact RBCs. Determining whether RMPs function in eCM pathogenesis represents a defined pilot RO3 research project with significant potential to provide preliminary data for a RO1 proposal. This proposal is significant because it will improve understanding and definition of CM pathogenesis (which is required to develop other adjunctive therapies aimed at protecting CM patients from the host response leading to death, cognitive impairment, and psychoses while the anti-parasite treatment kills Plasmodium falciparum), determine mechanisms of action of an adjunctive therapy undergoing clinical trials, and determine biomarkers for tracking NO bioavailability in malaria patients. Our group played a central role in defining that low NO bioavailability and microparticles are critical for eCM pathogenesis. We now extend our innovative research to link these 2 required mechanisms of pathogenesis. We hypothesize that RMPs play a critical role in the development of ECM by lowering nitric oxide (NO) bioavailability due to NO scavenging by hemoglobin. To address this hypothesis, we use the P. berghei- ANKA (PbA) infection of mice as a model for CM. Our data indicate that (i) low NO bioavailability is critical for eCM pathogenesis, (ii) MPs are critical for the development of eCM, and (iii) RMP numbers are markedly elevated in human CM and eCM. The contribution of RMPs to CM pathogenesis, however, remains to be determined. We therefore propose a limited RO3 project to test this hypothesis. In aim 1, we propose to analyze RMP's and unpackaged Hb's ability to scavenge NO in groups of (i) eCM-susceptible, (ii) eCM- resistant, eCM protected ([iii] inhaled NO and [iv] citrulline) mice infected with PbA and (v) eCM susceptible mice infected with non-eCM inducing P. berghei K173 (PbK). These studies determine the relative importance to low NO bioavailability during eCM of RMPs versus unpackaged Hb. In aim 2, we abrogate the production of ABCA1 in RBCs, which prevents the formation of MPs only in this cell type; comparison of survival of these mice to controls determines the importance of RMPs to eCM pathogenesis. We will also compare the pathogenic consequences of injecting equimolar doses of RMPs, unpackaged oxyHb, metHb, and intact RBCs into groups (i)-(v) of Aim 1 to assess the importance of the states of Hb to eCM pathogenesis. By comparing the results of our studies with eCM-protective NO therapy and vehicle control, we will not only define new mechanisms of action for inhaled NO and citrulline but also elucidate important pathological mechanisms in cerebral dysfunction that are amenable to treatment. These studies are significant because they directly address the pathogenic mechanisms of an important but often overlooked disease that kills millions of people and define the mechanisms of action of a potential therapy to rescue those presenting with cerebral malaria from their disease. Our team, comprising Dr. Gramaglia, Dr. Grau, Dr. Parks, and Dr. Torbett, has the expertise and research environment to perform the proposed studies.